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Issue with Xba1 and Dam methylation


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#1 Ahrenhase

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Posted 26 March 2013 - 04:19 PM

I'm cloning a few constructs into PCR2.1. One insert is flanked (inserted via PCR) by EcoR1 and Xba1, and the other is flanked by Xba1 sites on each side. Xba1 digestion is purportedly blocked by dam methylation; however, my first construct gets digested and my second one does not. How does that make sense?

#2 phage434

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Posted 26 March 2013 - 05:01 PM

DAM methylation occurs at GATC sites, where the A is methylated (the A on the reverse complement strand opposite the T also is methylated, its sequence is also GATC).

The XbaI site recognition site is TCTAGA. If your sequence is either GATCTAGA or TCTAGATC, then the GATC site will be methylated, and XbaI will not cut. Otherwise, it will.

#3 Ahrenhase

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Posted 26 March 2013 - 06:36 PM

DAM methylation occurs at GATC sites, where the A is methylated (the A on the reverse complement strand opposite the T also is methylated, its sequence is also GATC).

The XbaI site recognition site is TCTAGA. If your sequence is either GATCTAGA or TCTAGATC, then the GATC site will be methylated, and XbaI will not cut. Otherwise, it will.


Isn't the complement of TCTAGA - AGATCT, which contains GATC and is thus methylated?

#4 phage434

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Posted 26 March 2013 - 07:06 PM

No, you need the reverse complement, not the reverse. The reverse complement of TCTAGA is the same (TCTAGA).

#5 Ahrenhase

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Posted 27 March 2013 - 05:26 PM

No, you need the reverse complement, not the reverse. The reverse complement of TCTAGA is the same (TCTAGA).


Interesting, I didn't know RE's were directionally (5'->3') regulated, but I can see how that makes sense. That would explain why my bacterially-generated constructs are getting Xba1-digested.

#6 phage434

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Posted 27 March 2013 - 07:23 PM

I don't understand what you mean by "directionally regulated." They cut (typically) at palindromic sequences, such as GAATTC or TCTAGA. These sequences read the same in either direction (that is, they are their own reverse complements).

#7 Ahrenhase

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Posted 28 March 2013 - 02:03 AM

Sorry, what I meant was the Dam methylase, not the restriction enzyme. I was referring to the fact that 3'-GATC-5' is not methylated but 5'-GATC-3' is.

#8 phage434

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Posted 28 March 2013 - 03:15 AM

3' GATC 5' is utterly unrelated to 5' GATC 3' and it is not at all surprising that one is methylated and the other is not.
You need to deeply understand this.




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